Anti-obesity agent comprising compound containing benzotropolone ring

ABSTRACT

The object of the present invention is to provide an anti-obesity agent which contains a tea-derived component and which is safe and does not compromise the flavor of foods and beverages. 
     According to the present invention, a safe and palatable anti-obesity agent can be provided by incorporating a benzotropolone ring-containing compound which has tea-derived, high inhibitory activities against lipase and alfa-glucosidase. The anti-obesity agent of the present invention does not compromise the flavor of foods and beverage, has palatability, and can be used in various use applications including foods and beverages intended for health enhancement such as reduction in triglycerides.

CROSS REFERENCE TO RELATED APPLICATIONS:

This application is the National Stage of International Application No.PCT/JP2010/058624, filed May 21, 2010, and claims benefit of JapaneseApplication No. 2009-123585 filed May 21, 2009, which are hereinincorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to an anti-obesity agent containing abenzotropolone ring-containing compound.

BACKGROUND ART

Obesity is one of the most significant diseases in modern society andthe main factor of obesity is excessive intake of fat. Excessive intakeof fat is known to cause not only obesity but also diabetes,hyperlipidemia, hypertension, arteriosclerosis, and the like that areattributable to obesity. The condition in which two or more ofhyperglycemia, hypertension, and hyperlipidemia develop with visceralfat obesity is called metabolic syndrome (visceral fat syndrome), whichis at high risk of causing cardiac diseases and stroke and thus has beenregarded as a problem in recent years. As a therapeutic agent forobesity, for example, Xenical®, which has a suppressive action on fatabsorption from the gastrointestinal tract due to its lipase inhibitoryactivity, is commercially available as an anti-obesity agent; however,its side effects such as steatorrhea, increased frequency of defecation,loose stool, diarrhea, and stomachache have been reported and the agentthus cannot be necessarily safe (Non-Patent Document 1).

In order to prevent obesity, cutting calories on a restrictive diet isan effective way. Nevertheless, it is often hard to practice it in dailylife because substantial nutritional guidance has to be received.Accordingly, suppressing in a safe and healthy manner the absorption ofdiet-derived fat into the body is expected to be a realistic andeffective measure for treatment of obesity and obesity-related diseasesor health enhancement.

Under these circumstances, the development of specified health foodsthat are safe and proven effective in humans is attracting attention. Todate, the following food materials which suppress the elevation of serumtriglyceride level after meals are commercially marketed as specifiedhealth foods: globin digest which suppresses fat absorption by itspancreatic lipase inhibition; diacylglycerol, which has digestive andabsorptive properties different from triacylglycerol; eicosapentaenoicacid (EPA) and docosahexaenoic acid (DHA), which are purified from fishoil; and the like.

Also, recent interest is focusing on plant-derived lipase inhibitingactive substances, and particularly, the following polyphenols whichhave lipase inhibitory activity have been reported: plant bark-derivedtannin; tannins and flavonoids and glycosides thereof in the legumeCassia nomame; lipid absorption-inhibiting foods containingepigallocatechin gallate and epicatechin gallate, which are maincomponents in green tea; lipase inhibitors comprising water extractsfrom pepper, shimeji mushroom, pumpkin, maitake mushroom, seaweedHizikia fusiformis, green tea, oolong tea, and the like; flavones andflavonols; hydroxybenzoic acids (gallic acid), triterpene compounds andderivatives thereof; anti-obesity agents containing, as an activeingredient, procyanidin from tamarind; and the like. Further known arelipase inhibitory action of grape seed extract (Non-Patent Document 2);lipase inhibitory action from Salacia reticulate-derived polyphenol, andanti-obesity action in rats (Non-Patent Document 3); oolong teaextract-derived anti-obesity action in mice (Non-Patent Document 4); andthe like. In addition, teas contain a lot of catechins, many componentsof which have been separated and identified (Non-Patent Document 5), andthere are reports on lipase inhibitors containing tea-derived components(Patent Documents 1 and 2). Above all, theaflavins, known as pigments ofblack tea and oolong tea, exhibit strong lipase inhibitory activity inproportion to the number of gallate groups in a molecule (PatentDocument 2, Non-Patent Document 6). However, the content and proportionof these theaflavins are not constant among teas.

Alfa-glucosidase inhibiting substances have an inhibitory action on theelevation of blood glucose level by inhibiting alfa-glucosidase, whichis localized on small intestinal epithelium, and by suppressing ordelaying the decomposition and absorption of sugar. Accordingly,alfa-glucosidase-inhibiting substances are useful in various diseasessuch as diabetes and obesity, which are derived from the chronicity ofhigh blood sugar symptoms.

Since alfa-glucosidase inhibitory activity was discovered in maltcomponent in 1933, many alfa-glucosidase-inhibiting substances that arederived from wheat and pulse have been discovered. In 1966, nojirimycin,which has alfa-glucosidase inhibitory activity, was isolated from amicrobial metabolite and its structure was determined. From mulberryleaf extract, a related compound of nojirimycin, 1-deoxynojirimycin, wasobtained, which is known to have alfa-glucosidase inhibitory activity,and a method of extraction for keeping the activity from decreasing isdisclosed (Patent Document 3).

A compound that contains a 13-membered ring cyclitol structure having asulfoxide, which is isolated from the extract of the root of Salaciareticulate, is reported to have maltase inhibitory activity (PatentDocument 4). Diacylated pelargonidin, cyanidin, and peonidin3-sophoroside-5-glucosides are reported to have maltase inhibitoryactivity as an anthocyanin compound isolated from morning glories or theroot of purple sweet potato (Non-Patent Document 7). The maltaseinhibitory activity has also been confirmed in the components containedin tea leaves, such as theasinensin A, theaflavin derivatives having agalloyl group, and proanthocyanidins having epiafzelechingallate as aconstitutional unit. Although theaflavin derivatives having a galloylgroup have maltase inhibitory activity, they are contained in tea leavesin only a small proportion, 0.1 to 0.2% (Patent Document 5, Non-PatentDocument 8).

Black tea theaflavins and green tea catechins are reported to havealfa-glucosidase inhibitory activity (Non-Patent Document 8); theactivity has been confirmed in catechins having a galloyl group at their3 position including epigallocatechin-3-O-gallate (hereinafter referredto as “EGCG”) and epicatechin-3-O-gallate, and theaflavins includingtheaflavin-3-O-gallate and theaflavin-3,3′-di-O-gallate. The fractionsand the like of black tea have also been examined for theiralfa-glucosidase inhibitory activity; polymeric fractions formed byfermentation are also known to have the activity (Non-Patent Document9).

On the other hand, it is known that in fermentation process inproduction of black tea or oolong tea, polyphenols such as catechins orgallic acid are condensed into a compound having a benzotropolone ringthrough the activity of enzymes such as polyphenol oxidase in tea leaves(Non-Patent Document 10).

It is reported that aside from theaflavins, many benzotropolonering-containing compounds are present in teas. Reported are, forexample, apoptosis induction caused by purpurogallin derivatives (PatentDocument 6) and a method of manufacturing epitheaflagallins for use infoods (Patent Document 7), an enzymatic method of manufacturing atheaflavin type trimer, theadibenzotropolone A, and presence thereof inblack tea (Non-Patent Document 11), and the like. The anti-inflammatoryactions of various benzotropolone ring-containing compounds (Non-PatentDocument 12) are also known. Nevertheless, for benzotropolonering-containing compounds other than theaflavins and epitheaflagallins,nothing is known about their lipase inhibitory action relating to fatabsorption and their alfa-glucosidase inhibitory action relating totheir inhibitory action on the elevation of blood glucose level.

CITATION LIST Patent Document

-   -   Patent Document 1: WO2005/077384    -   Patent Document 2: WO 2006/004110    -   Patent Document 3: Japanese Patent Public Disclosure 2007-60908    -   Patent Document 4: Japanese Patent Public Disclosure 2008-137925    -   Patent Document 5: Japanese Patent Public Disclosure 2007-231009    -   Patent Document 6: Japanese Patent Public Disclosure 2004-359576    -   Patent Document 7: WO 2007-141945

Non-Patent Document

-   -   Non-Patent Document 1: Lancet 1998; 352: 167-172    -   Non-Patent Document 2: Nutrition 2003; 19(10): 876-879    -   Non-Patent Document 3: J. Nutr. 2002; 132: 1819-1824    -   Non-Patent Document 4: Int. J. Obes. 1999; 23: 98-105    -   Non-Patent Document 5: Journal of the Japanese Society for Food        Science and Technology, Vol. 46, No. 3, pp. 138-147, March 1999    -   Non-Patent Document 6: Chem. Pharm. Bull. 2008; 56: 266-272    -   Non-Patent Document 7: J. Agric. Food Chem.2001, 49, 1952-1956    -   Non-Patent Document 8: J. Agric. Food. Chem., 55, 99-105, 2007    -   Non-Patent Document 9: Chem. Pharm. Bull. 56(3), 266-272, 2008    -   Non-Patent Document 10: Tetrahedron 1973; 29: 125-142    -   Non-Patent Document 11: Tetrahedron Letters 2002; 43: 7129-7133    -   Non-Patent Document 12: Bioorganic & Medicinal Chemistry 2004;        12: 459-467

SUMMARY OF THE INVENTION Technical Problem

Even if some effect is found to be obtained from a plant extract, unlessthe quantity of the active components contained in the extract isdetermined, it is difficult to ensure stable maintenance of itsanti-obesity activity because the extract is of natural product origin.Further, some of the reported lipase inhibitors and alfa-glucosidaseinhibitors as shown above are not sufficiently effective.

A less palatable, vegetable-derived anti-obesity agent, when used asfoods or beverages, is expected to make a negative influence on theirflavor. On the other hand, a more palatable, tea-derived anti-obesityagent can possibly be an effective material candidate; however, forexample, even when we drink palatable black tea or oolong tea in orderto lower lipid, we cannot obtain its effect without drinking the tea inlarge amounts, and thus to practice it in daily life is thus notrealistic. Simply condensed black tea or oolong tea is also notappropriate to drink as a realistic measure because of its strongbitterness and astringency and increased caffein.

Accordingly, an object of the present invention is to provide ananti-obesity agent which is of natural product origin and effective.

Another object of the present invention is to provide a tea-derived,palatable inhibitor of lipase activity which shows a highly inhibitoryactivity against pancreatic lipase and suppresses absorption ofdiet-derived fat, and/or contributes to suppression and prevention ofobesity. Still another object is to provide an alfa-glucosidaseinhibitor which suppresses absorption of diet-derived sugar andcontributes to long-term prevention and/or treatment of diabetesresulting from the chronicity of high blood sugar symptoms.

Still another object of the present invention is to provide foods andbeverages which are palatable and intended to lower blood triglycerideand which enhance health.

Still another object of the present invention is to provide apharmaceutical composition which suppresses absorption of diet-derivedfat and elevation of blood triglyceride.

Solution to Problem

As a result of intensive studies to solve the above problems, thepresent inventors have found a component from tea leaves which potentlyinhibits pancreatic lipase, essential for fat absorption, and which hashigh alfa-glucosidase inhibitory activity. Specifically, the inventorshave assayed the inhibitory activities of various polyphenols present intea leaves against lipase and alfa-glucosidase, and found out that thebenzotropolone ring-containing compounds of Formula (1) have stronglipase inhibitory activity and strong alfa-glucosidase inhibitoryactivity. These compounds are oxides of catechins and polyphenols whichare contained in black tea, and thus are superior in flavor and safetyand can be taken for long periods of time. From these findings, theinventors have found that it is possible to provide foods and beverages,to which a lipase inhibitor and an alfa-glucosidase inhibitor are added,which are intended to suppress absorption of diet-derived fat and sugarand elevation of blood triglyceride and to prevent and/or treat diabetesresulting from the chronicity of high blood sugar symptoms is in thelong run. The present invention has been thus accomplished.

More specifically, the present invention is defined by [1] to [9] below.

[1] An anti-obesity agent comprising one or more compounds of Formula(1) (except epitheaflagallin, epitheaflagallin-3-O-gallate, theaflavin,theaflavin-3-O-gallate, theaflavin-3′-O-gallate, andtheaflavin-3,3′-O-digallate):

(wherein R₁ is H or OH;

R₂ is H or a group of Formula (2):

wherein R₄ is OH or a group of Formula (3):

or a group of Formula (4):

wherein R₃ is H, COOH, a group of Formula (5):

or a group of Formula (6):

wherein R₅ is OH, a group of Formula (7):

or a group of Formula (8):

wherein R₆ is a group of Formula (9):

or a group of Formula (10):

wherein R₁′ is the same group as R₁ above and R₂′ is a group of Formula(11):

wherein R₄′ is the same group as R₄ above).

[2] The anti-obesity agent according to [1], comprising one or morecompounds in which R₁ is H.

[3] The anti-obesity agent according to [1], wherein the compound is acompound of Formula (12):

[4] The anti-obesity agent according to any one of [1] to [3], which isa lipase inhibitor and/or an alfa-glucosidase inhibitor.

[5] The anti-obesity agent according to any one of [1] to [4], which isfor suppressing absorption of diet-derived fat and sugar.

[6] The anti-obesity agent according to any one of [1] to [5], which isin the form of a food or a beverage.

[7] The anti-obesity agent according to [6], wherein the food orbeverage is selected from the group consisting of a tea beverage, a softdrink, and a health food.

[8] The anti-obesity agent according to any one of [1] to [5], which isin the form of a pharmaceutical composition.

[9] A compound of Formula (24):

Advantageous Effects of the Invention

The lipase inhibitor that serves as the anti-obesity agent of thepresent invention contains a tea-derived benzotropolone ring-containingcompound and thus exhibits superior lipase inhibitory activity. Thelipase inhibitor of the present invention does not compromise the flavorof foods and beverages, has palatability, and can be used in various useapplications including foods and beverages intended for reduction intriglycerides and health enhancement. It is desirable to take thisinhibitor together with meals for suppression of dietary fat absorption,and therefore, beverages containing tea-derived, enhanced activeingredients are of great significance. Particularly, enhancing theseingredients enabled us to provide a beverage intended for anti-obesityaction and health enhancement.

The alfa-glucosidase inhibitor that serves as the anti-obesity agent ofthe present invention can suppress decomposition of sugar derived fromdiet-derived starch and polysaccharides and absorption of the sugar, inless amount than conventionally known, natural product-derivedalfa-glucosidase inhibitor. Further, all of the compounds are oxides ofcatechins and polyphenols contained in teas and are thus superior inflavor and safety, which enables long-term intake of the compounds.

In addition, the anti-obesity agent of the present invention contains abenzotropolone ring-containing compound, a component derived from teas,which are widely used in diet, and are thus safe and can be also used asa pharmaceutical composition with reduced side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ¹³C NMR spectrum of Compound 12.

FIG. 2 shows the ¹H NMR spectrum of Compound 12.

DESCRIPTION OF EMBODIMENTS

The embodiments of the present invention are described in detail below.

Anti-Obesity Agents

The present invention is an anti-obesity agent containing abenzotropolone ring-containing compound of Formula (1) as an activeingredient:

Particularly, the following is preferred as the active ingredient:purprogallin of Formula (12) below:

purprogallin carboxylic acid of Formula (13):

theaflavanin 3-O-gallate of Formula (14):

EGCG-catechol of Formula (15):

theaflavate A of Formula (16):

theadibenzotropolone A of Formula (17):

theaflavin digallate trimer 1 (TFdiGA-tri1) of Formula (18):

theaflavin digallate trimer 2 (TFdiGA-tri2) of Formula (19):

epitheaflavic acid of Formula (20):

3,4,6-trihydroxy-1-((2R,3R)-3,5,7-trihydroxychroman-2-yl)-5H-benzo[7]annulen-5-oneof Formula (21):

epitheaflavic acid-3-O-gallate of Formula (22):

or(2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychroman-3-yl2,3,4,6-tetrahydroxy-5-oxo-5H-benzo[7]annulen-8-carboxylateof Formula (23):

The above benzotropolone ring-containing compounds, which are activeingredients of the anti-obesity agent of the present invention, can beobtained by solvent extraction from natural materials such as green tea,black tea, and oolong tea or also by chemical synthesis or enzymaticsynthesis of the materials.

With respect to the natural materials which are extraction rawmaterials, tea leaves may be used as is or in ground form. The solventfor use in extraction may be water, an oraganic solvent, mixtures ofthese solvents, or the like, and preferably, hot water. The extractobtained can be separated and purified by using an adequate carrier forseparation. Any carrier can be used if the carrier can absorb the abovebenzotropolone ring-containing compound and separate it with an adequatesolvent for separation. For example, styrene- or dextran-based syntheticadsorbents can be used for the separation and purification. Afterloading of the above extract on such a carrier, an adequate solvent isused to separate the above benzotropolone ring-containing compound. Morespecifically, the above benzotropolone ring-containing compound for usein the anti-obesity agent of the present invention can be obtained inaccordance with the descriptions in Example 1 of the presentspecification. The thus obtained benzotropolone ring-containing compoundabove may be used in condensed form or as a powder obtained by methodssuch as lyophilization.

The above benzotropolone ring-containing compounds can be synthesizedwith enzymes such as polyphenol oxidase (PPO) and peroxidase (POD), orwith oxidants such as potassium ferricyanide (K₃[Fe(CN)₆]) as a catalystby using, as raw materials, catechins (epigallocatechin-3-O-gallate,epicatechin-3-O-gallate, epicatechin, epigallocatechin, and the like)and gallic acid. Specifically, the synthesis can be performed inaccordance with the descriptions in, for example, Examples 2 and 7 ofthe present specification.

The anti-obesity agent of the present invention has strong inhibitoryaction against lipase, especially, pancreatic lipase. The lipaseinhibitory activity can be measured by any method of the lipase activityassays described in the related applications shown in the Background Artsection. For example, the assay can be achieved by using the oleateester of fluorescent 4-methylumbelliferone as a substrate to measure thefluorescence of 4-methylumbelliferone produced by reaction with lipase.Exemplary is the method described in Example 6, which allows ameasurement of lipase inhibitory activity. Lipase inhibitory activitycan be expressed, for example, as IC₅₀, a sample volume which produces50% inhibition.

The anti-obesity agent of the present invention have potentalfa-glucosidase inhibitory activity. The activity can be measured byany method of the activity assays described in the related applicationsshown in the Background Art section. Exemplary is the method describedin Example 11, which allows a measurement of alfa-glucosidase inhibitoryactivity. Alfa-glucosidase inhibitory activity can be expressed, forexample, as IC₅₀, a sample volume which produces 50% inhibition.

The purified products or partially purified products of the abovebenzotropolone ring-containing compounds can be used alone as ananti-obesity agent, or can be combined with a solvent or a carrier so asto be used as an anti-obesity agent. It is preferred that the solvent orthe carrier can be used safely as a food or a pharmaceutical product, inprospect of use as the foods and beverages below and/or pharmaceuticalproducts. The anti-obesity agent of the present invention has varioususe applications; exemplary is use of the agent as foods and beveragesor pharmaceutical compositions which are used for an experimental studyand intended for prevention of triglyceride accumulation.

Foods and Beverages

The anti-obesity agent of the present invention may be in the form offood and beverage that suppresses undesirable elevation of bloodtriglyceride associated with fat intake from meals. Preferred examplesof the food and beverage include those taken on a daily basis; forexample, green tea, barley tea, oolong tea, black tea, coffee, sportsdrinks, drinking water, seasoning, and dressing. However, the foods andbeverages also may be those that are commonly consumed: soft drinks,cocktails, beer, whiskey, distilled spirit (shochu), wine, sake,seasoning, dressing, flavored rice, processed foods, instant foods,retort foods, chocolate, fresh cream, Western confectionery, dairyproducts, health foods, supplements, and the like.

In the anti-obesity agent of the present invention which is in the formof food and beverage, the benzotropolone ring-containing compound ofFormula (1) is contained so that the intake of the compound is 0.1 mg to10 g per meal, preferably, 0.5 mg to 5 g. However, the compound ofFormula (1) used in the anti-obesity agent of the present invention ishighly safe because it is derived from food, and thus there is nosubstantial upper limit on the amount of addition to foods andbeverages.

Pharmaceutical Composition

The anti-obesity agent of the present invention which serves as a lipaseinhibitor can also be used as a pharmaceutical composition intended tosuppress diet-derived fat absorption and to prevent undesirable increaseof blood triglyceride and/or to lower increased blood triglyceride. Theanti-obesity agent which serves as an alfa-glucosidase inhibitor canalso be used as a pharmaceutical composition intended to suppressdiet-derived fat absorption and to prevent undesirable increase of bloodtriglyceride and/or to lower increased blood triglyceride. Preferred areagents intended for oral administration; examples of the agent includedrinks, tablets, capsules, granules, powders, candies, drops, and thelike. The agents contain a benzotropolone ring-containing compound ofFormula (1) in amounts of 0.1 mg to 10 g per dose, preferably, 0.5 mg to5 g.

The pharmaceutical composition of the present invention is safe forlong-term dosage due to high safety of lipase and alfa-glucosidaseactivity of the inhibitory components. Accordingly, the composition canalso be taken on a daily basis to prevent or resolve obesity as alifestyle-related disease.

Novel Compound

Further, the present invention provides a compound shown below as anovel compound present in black tea.

This compound is useful as an anti-obesity material.

The present invention is more specifically described in Examples below,but is not limited thereto.

EXAMPLE 1

Separation on LH-20

Black tea leaves originated in Assam, India were extracted with hotwater at 90° C. and lyophilized, and distilled water was added to theresulting product followed by warming and dissolution to obtain asolution of concentration 10 mg/ml. While heating the solution to 60 to70° C. in a hot-water bath, 15 ml of the same was loaded on 60 ml ofSephadex LH-20 (GE Healthcare Biosciences, Ltd.) After washing with 45ml of 20% acetone (acetone:distilled water=2:8, v/v) and subsequentwashing with 60 ml (1 column volume) of the same, elution was conductedwith 4 column volumes each of the following solvents: 30% acetone(acetone:distilled water=3:7, v/v); 50% acetone (acetone:distilledwater=1:1, v/v); and 60% acetone (acetone:distilled water=6:4, v/v), andthe resulting solution was fractionated into 1 column volume for eachfraction. Two milliliters each taken from a total of fourteen 60 mlfractions was concentrated under vacuum and then 0.1 ml of a 50% aqueousdimethylsulfoxide solution (dimethylsulfoxide:distilled water=1:1, v/v)was prepared, and the solution was subjected to a measurement of lipaseinhibitory activity. In addition, each fraction was concentrated undervacuum and lyophilized, and the recovery volume was calculated from theresulting solids; the calculations were used for the component analysisconducted in Example 5. The weights and the recovery rates of lipaseinhibitory activity are shown in Table 1.

TABLE 1 Location of Activity in LH20 Fractions Abundance Abundance Ratioof Ratio of Yield Fra No. Fraction Activity (%) (%) 1 20% acetone_10.0005 5.0 30.6 54.3 2 20% acetone_2 5.01 23.7 3 30% acetone_1 4.72 20.13.7 16.3 4 30% acetone_2 6.30 4.7 5 30% acetone_3 5.63 5.1 6 30%acetone_4 3.48 2.8 7 50% acetone_1 14.12 70.9 4.8 23.6 8 50% acetone_233.59 11.2 9 50% acetone _3 17.08 5.1 10 50% acetone_4 6.15 2.5 11 60%acetone_1 2.53 3.9 1.3 5.8 12 60% acetone_2 1.05 1.8 13 60% acetone_30.20 1.2 14 60% acetone_4 0.13 1.5

As a result of the measurement of lipase inhibitory activity inaccordance with the method described in Example 6, 20% acetone_(—)1 and20% acetone_(—)2 fractions, which exhibited high yield, were each foundto have contained polysaccharides and caffeins and have had almost noactivity. On the other hand, in 50% acetone_(—)2 fraction, whichexhibited the next highest yield (11.2% by weight), activity of 33.6%was eluted; in the subsequent entry, 50% acetone_(—)3 fraction, activityof 17.1% was eluted. In the 50% acetone elution portions including thesefractions, it was found that 70.9% of the activity was eluted. In theassay described in Example 5, many of active benzotropolonering-containing compounds were eluted with 50% acetone, which is wellconsistent with the high activity found in 50% acetone elution portions.

EXAMPLE 2

Syntheses of Test Compounds

-   1. purprogallin-   2. purprogallin carboxylic acid-   3. epitheaflagallin-   4. epitheaflagallin-3-O-gallate-   5. theaflavate A-   6. theadibenzotropolone A-   7. theaflavin digallate trimer 1 (TFdiGA-tri1)-   8. theaflavin digallate trimer 2 (TFdiGA-tri2)-   9. epitheaflavic acid-   10. theaflavanin-   11. epitheaflavic acid-3-O-gallate-   12.    (2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychroman-3-yl2,3,4,6-tetrahydroxy-5-oxo-5H-benzo[7]annulen-8-carboxylate

Syntheses of epitheaflagallin (3) and epitheaflagallin-3-O-gallate (4)

To epigallocatechin (0.2 mmol) (EGC; Wako Pure Chemical Industries,Ltd.) or epigallocatechin-3-O-gallate (0.2 mmol) (EGCG; Wako PureChemical Industries, Ltd.), potassium ferricyanide (0.8 mmol) and NaHCO₃(0.8 mmol) were added to prepare 150 ml of an aqueous solution, and thesolution was chilled on ice. Fifty milliliters of an aqueous pyrogallolsolution (0.2 mmol) was dripped into the solution and stirring themixture were continued for one hour. The reaction solution was loaded on20 ml volume of Sep-pak C18 (Waters Corp.), and after washing with 60 mlof water and the subsequent washing with 5% acetonitrile/water, 50%acetonitrile/water containing 0.1% formic acid was used to elute areaction product. The eluted product was lyophilized and purified bypreparative HPLC shown below.

The reaction product of EGC and pyrogallol was loaded on YMC Pak PolymerC-18 (20×300 mm, YMC Co., Ltd.) and an elution with a linear gradient of30-50% acetonitrile (6 ml/min, 60 minutes) was performed in the presenceof 0.1% formic acid. The component eluted at between 48 and 50 minuteswas lyophilized to obtain 4 mg of a brown solid (epitheaflagallin (3)).Also, the component eluted at between 68 and 70 minutes in thischromatogram was lyophilized to obtain 2 mg of a brown solid(purpurogallin (1)).

The reaction product of EGCG and pyrogallol was loaded on DevelosilC30-UG-5 (20 mm×250 mm, Nomura Chemical Co., Ltd.) and an elution with alinear gradient of 15-50% acetonitrile (6 ml/min, 60 minutes) wasperformed in the presence of 0.1% formic acid. The component eluted atbetween 44 and 46 minutes was lyophilized to obtain 6 mg of a brownsolid (epitheaflagallin-3-O-gallate (4)). Also, the component eluted atbetween 48 and 50 minutes in this chromatogram was lyophilized to obtain3 mg of a brown solid (purpurogallin (1)).

In the same manner as the above reactions, 800 mg of gallic acid wassubjected to a reaction to obtain 65 mg of purprogallin carboxylic acid(2), which is produced by polymerization of two gallic acid molecules.In addition, 100 mg of epicatechin-3-O-gallate (ECG) was subjected to areaction to obtain 3 mg of theaflavate A (5), which is produced bypolymerization of two ECG molecules. Further, 400 mg each of epicatechin(EC) and EGCG were subjected to a reaction to obtain a main product,theaflavin-3-O-gallate as well as 2 mg of a by-product,theadibenzotropolone A (6). In the same manner as the above reaction,200 mg each of epicatechin-3-O-gallate (ECG) and EGCG were subjected toa reaction, and 12 mg of the resulting theaflavin-3,3′-digallate and 20mg of ECG were subjected to a reaction to obtain 1.7 mg of TFdiGA-tri1(7) and 0.6 mg of TFdiGA-tri2 (8). The following compounds were alsoobtained: 48 mg of epitheaflavic acid (9) obtained by reaction of 330 mgof gallic acid and 101 mg of EC; 12 mg of theaflavanin (10) obtained byreaction of 869 mg of pyrogallol and 400 mg of EC; and 37 mg ofepitheaflavic acid-3-O-gallate (11) and 3.3 mg of(2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychroman-3-yl2,3,4,6-tetrahydroxy-5-oxo-5H-benzo[7]annulen-8-carboxylate(12) which were simultaneously obtained by reaction of 470 mg of gallicacid and 221 mg of ECG.

EXAMPLE 3

Structural Analyses of Compounds

MS and NMR measurements of the compounds obtained in Example 2 wereperformed. Their mass spectra were determined with Q-TOF Premier(Micromass Co., Ltd., UK) using Z-Spray ESI ion source, in negative, Vmode. Cone voltage: 45V, Capillary voltage: 3 KV, Source Temp.: 80° C.,Desolvation Temp.: 180° C. Mass correction was performed with LockSpray,and leucine enkepharine (m/z 554.2615 [M-H]−) was used as a reference.

The accurate mass, molecular formula, and theoretical mass value of eachcompound are shown in Table 2.

TABLE 2 Mass Spectrometric Results of Compounds Compound m/z ActualMolecular ma/z Calc. No. Compound Meas. Value Formula Value 1Purprogallin 219.0285 C11H8O5 219.0293 2 purprogallin carboxylic acid263.0183 C12H8O7 263.0192 3 epitheaflagallin 399.0701 C20H16O9 399.07164 epitheaflagallin-3-O-gallate 551.0818 C27H20O13 551.0826 5theaflavateA 851.1471 C43H32O19 851.1460 6 theadibenzotropoloneA973.1832 C50H38O21 973.1827 7 TFdiGA-tri1 1279.2185 C64H46O29 1279.22038 TFdiGA-tri2 1279.2218 C64H46O29 1279.2203 9 epitheaflavic acid427.0666 C21H16O10 427.0665 10 theaflavanin 383.0764 C20H16O8 383.076711 epitheaflavic acid-3-O-gallate 579.0778 C28H20O14 579.0775 12(2R,3R)-2-(3,4-dihydroxyphenyl)- 535.0883 C27H20O12 535.08775,7-dihydroxychroman-3-yl 2,3,4,6- tetrahydroxy-5-oxo-5H-benzo[7]annulene-8-carboxylate 14 theaflavanin-3-O-gallate 535.0871C27H20O12 535.0877 15 EGCG-catechol 535.0885 C27H20O12 535.0877

EXAMPLE 4

(Quantification in Tea Leaf Extract)

The LC-MS/MS measurements were performed with 4000 Q TRAP (AppliedBiosystems Inc.) using TurboIonSpray in negative mode under thefollowing conditions: Collision energy: 46 eV (nega.); Ionspray voltage:4500V; Temperature: 450° C.

The elution time and measurement channels of each compound in MRM(multiple reaction monitoring) mode are as shown in Table 3.

TABLE 3 LC-MSMS Quantitative P arameters Compound Compound R.T. Q1/Q3 1purprogallin 10.99 219.0/191.0 2 purprogallin carboxylic acid 8.01263.0/190.9 3 epitheaflagallin 8.64 399.1/233.0 4epitheaflagallin-3-O-gallate 11.54 551.1/233.1 5 theaflavateA 14.46851.2/579.1 6 theadibenzotropoloneA 11.49 973.2/227.1 7 TFdiGA-tri115.01 1277.2/579.1  8 TFdiGA-tri2 17.37 1277.2/697.1 

Column: YMC-Polymer C18, S-6 μm (YMC Co., Ltd., 2 mmφ×150 mm)

Flow Rate: 0.2 mL/min

Column Temp.: 40° C.

Mobile Phase A: 0.1V/V % HCOOH/H₂O

Mobile Phase B: 0.1V/V % HCOOH/CH₃CN

Gradient Program: A/B=91/9 (0 min)→A/B=40/60 (17 min)→A/B=15/85 (17.1min)→A/B=15/85 (17.1 to 19 min)

The above conditions were used to assay a hot water extract of IndianAssam CTC tea. Compounds 2 to 8 were found to have been present in thetea leaves. The quantitative values are shown in Table 4.

TABLE 4 Quantitative Values of Black Tea Components Compound Compoun μg/g extract 1 purprogallin 0.15 2 purprogallin carboxylic acid 166.00 3epitheaflagallin 224.00 4 epitheaflagallin-3-O-gallate 369.0 5theaflavateA 165.5 6 theadibenzotropoloneA 35.95 7 TFdiGA-tri1 67.20 8TFdiGA-tri2 61.55

EXAMPLE 5

(Distribution of Each Compound in Tea Leaf Fraction)

LC-MS/MS measurements were performed with Q-TOF Premier (Micromass Co.,Ltd., UK) using Z-Spray ESI ion source, in negative, V mode. Conevoltage: 33V, Capillary voltage: 3 KV, Source Temp.: 150° C.,Desolvation Temp.: 250° C. Mass correction was performed with LockSpray,and leucine enkepharine (m/z 554.2615 [M-H]⁻) was used as a reference.

Column: YMC-Polymer C18, S-6 μm (YMC Co., Ltd., 2 mmφ×150 mm)

Flow Rate: 0.2 mL/min

Column Temp.: 40° C.

Mobile Phase A: 0.1V/V % HCOOH/H₂O

Mobile Phase B: 0.1V/V % HCOOH/CH₃CN

Gradient Program: A/B=70/30→A/B=50/50 (20 min)→A/B=50/50 (25 min)

The above conditions were used to analyze each of the fractions of tealeaves originated in Assam, India, which were obtained by fractionationin Example 1. Compound 2 (m/z 263.02, R.T.=9.29 min) was detected in 30%acetone_(—)1 fraction, Compound 3 (m/z 399.07, R.T.=9.83 min) was in 50%acetone_(—)2 and 50% acetone_(—)3 fractions, Compound 4 (m/z 551.08,R.T.=13.48 min) was in 50% acetone_(—)3 and 50% acetone_(—)4 fractions,Compound 5 (m/z 851.15, R.T.=17.50 min) was in 50% acetone_(—)4fraction, Compound 9 (m/z 427.07, R.T.=8.60 min) was in 50% acetone_(—)3fraction, Compound 10 (m/z 383.08, R.T.=9.65 min) was in 30%acetone_(—)3 and 30% acetone_(—)4 fractions, Compound 11 (m/z 579.08,R.T.=13.86 min) was in 50% acetone_(—)2 fraction, and Compound 12 (m/z535.09, R.T.=13.90 min) was in 50% acetone_(—)2 fraction.

EXAMPLE 6

Measurement of Lipase Inhibitory Activity

Measurement samples were synthesized and purified in accordance with themethod in Example 2.

-   1. purprogallin-   2. purprogallin carboxylic acid-   5. theaflavate A-   6. theadibenzotropolone A-   7. theaflavin digallate trimer 1 (TFdiGA-tri1)-   8. theaflavin digallate trimer 2 (TFdiGA-tri2)-   9. epitheaflavic acid-   10. theaflavanin-   11. epitheaflavic acid-3-O-gallate-   12.    (2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychroman-3-yl2,3,4,6-tetrahydroxy-5-oxo-5H-benzo[7]annulen-8-carboxylate-   13. EGCG (Wako Pure Chemical Industries, Ltd.)

Method of Measurement

The measurement of lipase activity was performed by using as a substratethe oleate ester of fluorescent 4-methylumbelliferone (4-MUO;Sigma-Aldrich Corp.) to measure the fluorescence of4-methylumbelliferone produced by a reaction. In the measurement, 13 mMTris-HCl (pH8.0) containing 150 mM NaCl and 1.36 mM CaCl₂ was used as abuffer. The following were subjected to an enzymatic measurement: thesubstrate 4-MUO which was dissolved into a 0.1M DMSO solution followedby dilution of the solution 4000-fold with the above buffer; and as alipase, porcine pancreatic lipase (Sigma-Aldrich Corp.) which wasprepared as a 400 U/ml solution by using the above buffer likewise.

An enzymatic reaction was initiated by the following steps under 25° C.condition: adding to a 96-well microplate 50 μl of a 4-MUO buffersolution and 25 μl of distilled water (or an aqueous sample solution)for each well; mixing them; and then adding 25 μl of a lipase buffersolution to each well. After a 30-minute reaction, 100 μl of a 0.1Mcitric acid buffer (pH4.2) was added to terminate the reaction, and thefluorescence of 4-methylumbelliferone (excitation wavelength: 355 nm,fluorescence wavelength: 460 nm) produced by the reaction was measuredwith a fluorescence plate reader (Fluoroskan Asent CF from Labsystems,Inc.)

The inhibitory activity of test samples were determined as IC₅₀ (μM), asample volume which produces 50% inhibition, relative to the activity ofa control (distilled water).

Results

The lipase inhibitory activity of Compounds 1, 2, and 5 to 14 is shownin Table 5.

TABLE 5 Lipase Inhibitory Activity per mole Compound IC50 No. CompoundμM — pyrogallol 17.273 1 purprogallin 0.413 2 purprogallin carboxylicacid 4.165 5 theaflavateA 0.202 6 theadibenzotropoloneA 0.178 7TFdiGA-tri1 0.135 8 TFdiGA-tri2 0.138 9 epitheaflavic acid 4.626 10theaflavanin 0.984 11 epitheaflavic acid 3-O-gallate 0.266 12(2R,3R)-2-(3,4-dihydroxyphenyl)- 0.207 5,7-dihydroxychroman-3-yl2,3,4,6-tetrahydroxy-5-oxo-5H- benzo[7]annulene-8-carboxylate 13 EGCG0.349

All of the benzotropolone ring-containing compounds exhibited lipaseinhibitory activity. In (−)-epigallocatechin-3-O-gallate (EGCG; Compound13), which is known to exhibit strong lipase inhibition, its IC₅₀ was0.349 μM. The compounds other than purprogallin carboxylic acid (2) andepitheaflavic acid (9) which have a free carboxylic acid residueexhibited activity equivalent to or stronger than EGCG, which showedthat their benzotropolone ring contributes to their lipase inhibitoryactivity. A carboxylic acid residue was found to serve to reduce theactivity. Accordingly, in Formula (1), R₃ is preferably a group otherthan COOH.

EXAMPLE 7

(1) Preparations of Compounds

Syntheses of Compounds 12 and 14 (theaflavanin 3-O-gallate) withPeroxidase

Used were horseradish peroxidase from Zymed Laboratories, Inc. as aperoxidase, epicatechin 3-O-gallate (ECG) of 90% purity or higher, whichwas purified by reversed-phase HPLC from tea extract, and pyrogallolfrom Nacalai Tesque, Inc. (99.0% purity).

(2) Reactions

In 10 ml of a 0.058 M acetic acid buffer, 4.3 mg of the horseradishperoxidase was dissolved, and to this solution, 250 mg of ECG (0.566mmol) dissolved in 500 μl of acetone and 192.8 mg of pyrogallol (1.53mmol) dissolved in 500 μl of acetone were added followed by stirring.Under 30° C. condition, 450 μl of a 3% (w/v) hydrogen peroxide solutionwas added to initiate a reaction. For improvement of reactionefficiency, 450 μl of a 3% (w/v) hydrogen peroxide solution was addedtwice, i.e., after 10 and 20 minutes of the reaction initiation. Addedwere 192.8 mg of pyrogallol (1.53 mmol) and 450 μl of a 3% hydrogenperoxide solution after 30 minutes of the reaction initiation, and thenreacted for another 30 minutes.

After 60 minutes of the reaction initiation, the reaction solution wasloaded on a reversed-phase stationary phase (Waters Corp., Sep-Pak,C18-Vac 20 cc (5 g)) followed by washing with 40 ml of distilled water.Consecutive elutions were then performed with 20 ml of a 20% (v/v)aqueous acetonitrile solution and then with 40 ml of a 70% (v/v) aqueousacetonitrile solution. The 70% acetonitrile eluate was concentrated andlyophilized to obtain 68.0 mg of a fraction containing Compounds 12 and14 (theaflavanin 3-O-gallate).

The mixture containing Compounds 12 and 14 (theaflavanin 3-O-gallate)was purified by HPLC under the conditions below.

The mixture was loaded on YMC-Pak Polymer C-18 (20×300 mm, YMC Co.,Ltd.), and in the presence of 0.1% formic acid, a 30-minute isocraticelution with 30% acetonitrile and then an elution with a linear gradientof 30-45% acetonitrile (6 ml/min, 150 minutes) were performed. Thecomponent eluted at between 144 and 148 minutes and that eluted atbetween 158 and 162 minutes were lyophilized to obtain 3.9 mg of thecompound identical to Compound 12 shown in Example 2 and 3.0 mg ofCompound 14 (theaflavanin 3-O-gallate). Further, the component eluted atbetween 108 and 113 minutes in this chromatogram was lyophilized toobtain 36 mg of a brown solid (Compound 1 in Example 2: purprogallin).

EXAMPLE 8

Instrumental and Structural Analyses of Reaction Products

Compounds 12 and 14, which were obtained in Example 7, were subjected toMS and NMR measurements.

Their mass spectra were determined with Q-TOF Premier (Micromass Co.,Ltd., UK) using Z-Spray ESI ion source, in negative, V mode. Cone volt.:33 V, Capillary voltage: 2.7 kV, Source Temp.: 80° C., DesolvationTemp.: 180° C. Mass correction was performed with LockSpray, and leucineenkepharine (m/z 554.2615 [M-H]⁻) was used as a reference. The collisionenergy was set at 4 eV at the time of MS measurement and at 22 eV at thetime of MS/MS measurement.

Compounds 12 and 14 produced molecular ions of m/z 535.0883 and 535.0871[M-H]−, respectively, and their molecular formula was determined asC₂₇H₂₀O₁₂ (theoretical value: 535.0877). As a result of the MS/MSmeasurement in which collision energy was set at 22 eV, fragment ions of263.07 and 219.07 were detected in Compound 12 and those of 383.08 and169.01 were in Compound 14. Compound 14 was found to be a knowncompound, theaflavanin 3-O-gallate.

An NMR measurement was performed to confirm the structure of Compound12. The ¹³C NMR and ¹H NMR spectra of Compound 12 are shown in FIGS. 1and 2, respectively. The NMR measurement was performed under theconditions below. In CD₃OH, 3 mg of Compound 12 obtained in Example 7was dissolved, and the residual peaks of protons and ¹³C of CD₃OH, δ3.30and δ48.97, were set as internal standards. Measurements in accordancewith the following methods were performed with a DMX-750 spectrometer(BRUKER BIOSPIN, Germany): ¹H NMR, ¹³C NMR, ¹H{¹³C}-HSQC, ¹H{¹³C}-HMBC,TOCSY, DQF-COSY, NOESY, and ROESY. As a result, Compound 12, which wasobtained in Example 7, was confirmed to have the structure shown below.

The numbering of each atom is shown in the above structural formula.

The NMR measurement results and the signal assignments are shown below.

TABLE 6 ¹H ¹³C δ J (Hz) δ A(C)-ring C-2 5.09 brs 78.23 C-3 5.58 dd 71.75C-4 2.94 dd 17.4, 1.9 26.48 A-5 3.04 dd 17.4, 4.5 157.01 A-6 6.02 d  296.75 A-7 OH 157.91 A-8 5.97 d  2 95.8 A-8a 158.07 C-4a 99.07 B-ring B-1131.25 B-2 6.95 d  1.4 114.76 B-3 OH 146.25 B-4 OH 146.11 B-5 6.72 d  8116.04 B-6 6.83 dd  8, 1.4 118.87 benzotropolon a C═O 184.35 b OH 154.63c 7.51 d  1 114.45 d 125.12 e 8.03 brs 139.62 f 6.91 s 114.76 g OH152.62 h OH 138.44 i OH 153.73 j 116.29 k 131.96

The structure of Compound 14 is also shown below.

EXAMPLE 9

Synthesis of Compound 15

To 229.2 mg of epigallocatechin-3-O-gallate (0.5 mmol) (EGCG; Wako PureChemical Industries, Ltd.), 3.293 g of potassium ferricyanide (10 mmol)(Nacalai Tesque, Inc.) and 0.84 g of NaHCO₃ (10 mmol) were added toprepare 400 ml of an aqueous solution, and the solution was chilled onice. Into the solution, 100 ml of an aqueous solution of 275.3 mg ofcatechol (2.5 mmol) was dripped over one hour, and the mixture was keptstirred. The reaction solution was loaded on 300 mL of Sephadex LH-20(GE Healthcare Biosciences, Ltd.) and elutions were performed with 1 Lof 40% acetone/water, 1.2 L of 45% acetone/water, and 900 mL of 50%acetone/water, consecutively, followed by lyophilization. Consequently,77 mg of a 45% fraction containing EGCG-catechol was obtained. Thefraction was purified by preparative HPLC shown below.

The fraction containing EGCG-catechol was loaded on YMC-Pak Polymer C-18(20×300 mm, YMC Co., Ltd.), and at a flow rate of 6 ml/min in thepresence of 0.1% formic acid, an elution with a linear gradient of25-45% acetonitrile (75 minutes) was performed, and then a 30-minuteisocratic elution was maintained with 45% acetonitrile. The componenteluted at between 92 and 95 minutes was lyophilized to obtain 24 mg of abrown solid (Compound 15: EGCG-catechol). The structure of Compound 15is shown below.

EXAMPLE 10

Measurement of Lipase Activity

Method of Measurement

The measurement of lipase activity was performed by using as a substratethe oleate ester of fluorescent 4-methylumbelliferone (4-MUO;Sigma-Aldrich Corp.) to measure the fluorescence of4-methylumbelliferone produced by a reaction. In the measurement, 13 mMTris-HCl (pH8.0) containing 150 mM NaCl and 1.36 mM CaCl₂ was used as abuffer. The following were subjected to an enzymatic measurement: thesubstrate 4-MUO which was dissolved into a 0.01M DMSO solution followedby dilution of the solution 667-fold with the above buffer; and as alipase, porcine pancreatic lipase (Sigma-Aldrich Corp.: Type VI-S) whichwas prepared as a 400 U/ml solution by using the above buffer likewise.

An enzymatic reaction was initiated by the following steps under 25° C.condition: adding to a 96-well microplate 50 μl of 4-MUO buffer solutionand 25 μl of distilled water (or aqueous sample solution) for each well;mixing them; and then adding 25 μl of a lipase buffer solution to eachwell. This measurement was designed to increase the solubility of thesubstrate to enable inhibitory activity to be measured more accurately,because the concentration of the substrate was 7.5 μM at the time of thereaction, which showed its dilution as compared with the concentration12.5 μM achieved in Example 6 and the DMSO concentration was increased.After a 30-minute reaction, 100 μl of 0.1M citric acid buffer (pH4.2)was added to terminate the reaction, and the fluorescence of4-methylumbelliferone (excitation wavelength: 355 nm, fluorescencewavelength: 460 nm) produced by the reaction was measured with afluorescence plate reader (Fluoroskan Asent CF from Labsystems, Inc.).

The inhibitory activities of test samples were determined as IC₅₀ (μM),a sample volume which produces 50% inhibition, relative to the activityof a control (distilled water).

The lipase inhibitory activity of Compounds 3, 4, 12, 13, 14, and 15 wasmeasured. The results are shown in Table 7.

TABLE 7 Lipase Inhibitory Activity per Mole IC50 Compound Compound μM 3epitheaflagalin 0.956 4 epitheaflagalin 3-O-gallate 0.125 12(2R,3R)-2-(3,4-dihydroxyphenyl)- 0.096 5,7-dihydroxychroman-3-yl2,3,4,6-tetrahydroxy-5-oxo-5H- benzo[7]annulene-8-carboxylate 13 EGCG0.349 14 theaflavanin 3-O-gallate 0.168 15 EGCG-catechol 0.104

Compounds 12, 14, and 15 all had stronger activity than a positivecontrol, EGCG, and exhibited activity equivalent to or stronger thanCompound 4 (Japanese Patent Public Disclosure 2009-114079), which isknown as a lipase inhibitor.

The structures of Compounds 14 and 15 are shown as antioxidants andanti-inflammatory pharmaceutical agents in Japanese Patent PublicDisclosure 2007-504168, but their lipase and alfa-glucosidaseinhibitions were not known.

Compounds 12 and 14 can be synthesized by using polyphenol oxidase (PPO)or oxidants such as potassium ferricyanide besides the enzyme shown inExamples. Also, the compounds can be synthesized not only by acombination of ECG and pyrogallol but also by reaction with gallic acid.

EXAMPLE 11

Measurement of Alfa-Glucosidase Inhibitory Activity of VariousBenzotropolone Ring-containing Compounds

A 1M sodium phosphate buffer was prepared by mixing a 0.1M NaH₂PO₄-2H₂Oand a 0.1M Na₂HPO₄-12H₂O and adjusting the mixture to pH7.0, and thereto2 g/L of bovine serum albumin (Nacalai Tesque, Inc., F-V, pH5.2, 96%purity) and 0.2 g/L of NaN₃ (Nacalai Tesque, Inc., a special gradereagent) were added. To prepare an enzyme solution, alfa-glucosidase(Wako Pure Chemical Industries, Ltd., yeast-derived, 100 units/mg) wasdissolved in the above buffer so as to achieve 0.5 units/mg protein/ml(100 μmg/20 ml). To prepare a substrate solution,p-nitrophenyl-alfa-D-glucopyranoside (Nacalai Tesque, Inc., a specialgrade reagent) was dissolved in the above buffer so as to achieve 5 mMconcentration (7.525 mg/5 ml).

Among the samples used for the assays, epigallocatechin-3-O-gallate(Compound 13: EGCG), a positive control, was a product from Wako PureChemical Industries, Ltd., and Compounds 1, 3, 4, 5, 11, 12, 14, and 15were products that were synthesized and purified in Example 1, 2, or 7.

These samples were adjusted so as to obtain 10 mg/ml of DMSO and thesolution was diluted 2-fold in 6 steps. Using a 96-well microplate, 45μL of the enzyme solution was added to 10 μL of the sample solution.After preincubation at 37° C. for 5 minutes, 45 μL of the substratesolution was added and absorbance at 405 nm (A405 nm) was measured.After incubation at 37° C. for 5 minutes, the absorbance A405 nm wasmeasured. Percent inhibition was calculated as a difference in A405 nmfrom a solution in which only DMSO was added as a control instead of asample, and two consecutive measurements were performed for activity ofeach compound.

As a result, the alfa-glucosidase inhibitory activity of each Compound,when indicated by IC₅₀ value, is as shown in Table 8; Compounds 12 and14 exhibited particularly strong activity.

TABLE 8 alfa-glucosidase inhibitory activity IC50 Compound Compound (mM)1 purpurogallin 1.237 3 epitheaflagallin No 4 epitheaflagallin3-O-gallate 0.180 5 theaflavate A 0.116 11 epitheaflavic acid-GA 0.28712 (2R,3R)-2-(3,4-dihydroxyphenyl)- 0.159 5,7-dihydroxychroman-3-yl2,3,4,6-tetrahydroxy-5-oxo-5H- benzo[7]annulene-8-carboxylate 13 EGCG0.485 14 theaflavanin 3-O-gallate 0.196 15 EGCG-catechol 0.103

From these results, together with the results on lipase inhibitoryactivity, these benzotropolone ring-containing compounds were found tohave strong inhibitory activities against digestive enzymes. Among all,Compound 12, which exhibited its strong inhibitory activities againstboth the two enzymes, was confirmed to be present also in black tea; todate, the compound has not been known, but was found to be useful as ananti-obesity material.

Industrial Applicability

The anti-obesity agent of the present invention contains a tea-derivedbenzotropolone ring-containing compound and thus exhibits superiorinhibitory activities against lipase and alfa-glucosidase. The agentdoes not compromise the flavor of foods and beverages, has palatability,and can be used in various use applications including foods andbeverages intended for health enhancement such as reduction intriglycerides.

The invention claimed is:
 1. A compound of Formula (24):


2. A composition comprising the compound of claim
 1. 3. A food or abeverage comprising the compound of claim
 1. 4. The food or beverage ofclaim 3, wherein the food or beverage is selected from the groupconsisting of a tea beverage, a soft drink, and a health food.